Proton (H+, hydrogen ion)-induced bone resorption is common during renal osteodystrophy and has been implicated as a factor in the development of osteoporosis. The studies outlined here will increase our understanding of the mechanisms by which osteoblasts detect changes in [H+] and the signaling events that ultimately lead to H+-induced, cell-mediated bone resorption. We and others have shown that recently identified G-protein coupled proton receptors (PSR) are present in osteoblasts which respond to an increase in H+ with a transient increase in intracellular Ca (Cai). We have also shown that in cultured bone, an increase in H+ induces a prostaglandin-dependent increase in osteoblastic RANKL leading to enhanced bone resorption. The goal of this proposal is to link the activation of a PSR expressed in osteoblasts, OGR1, to H+-induced, cell-mediated bone resorption. We propose to test 4 specific hypotheses: 1) That specific H+- induced intracellular signaling in the osteoblast is mediated by the PSR, OGR1. We will determine the abundance and distribution of OGR1 at various stages of bone development and in primary neonatal calvarial osteoblasts and whether signaling via H+ activation of PSR(s) in osteoblasts parallels signal transduction pathways activated by H+ in a cell line expressing recombinant OGR1;2) That H+ activation of OGR-1 in osteoblasts is necessary for H+-induced, cell-mediated bone resorption by generating a transgenic mouse strain with an osteoblast specific chromosomal deletion of OGR1 using homologous recombination in mouse embryonic stem cells. This loss of function mutation will be characterized by bone morphology as well as the systemic response of these mice to H+. We will measure H+ effects on intracellular signaling, COX2 and RANKL expression in primary calvarial cells and subsequent bone resorption in cultured calvariae;3) That activation of OGR1 by H+ initiates specific intracellular signal transduction pathways in the osteoblast required for subsequent bone resorption. We will determine the role of p38 MAP kinase in regulating osteoblastic COX2 expression and subsequent stimulation of RANKL. We will identify specific sequence elements within the RANKL and COX2 promoters required for their H+- induction;4) That the concentration of bicarbonate ([HCO3-]) modulates the response of OGR1 to H+. Improving our understanding of the initial signaling events in the response of osteoblasts to H+ will aid in devising strategies to preserve bone during acidosis while maintaining its important H+ buffering properties. Lay Language Summary - Our bodies make acid that must be neutralized or it will have an adverse affect on our health. Bone neutralizes the acid but, in doing so, loses calcium and other minerals. The current proposal is aimed at understanding the first steps by which acid is detected by bone cells. Understanding the process by which the bone detects and neutralizes the acid can lead to an improvement in bone health.